For our first specific aim, we have created a series of transgenic mice which overexpress methionine sulfoxide reductase in specific cellular locations. We have begun studies of cultured fibroblasts from these animals and have initiated a collaborative effort to determine the life span of these animals to test the hypothesis that increased activity will extend lifespan. To directly test the hypothesis that methionine in proteins functions as a protective antioxidant, we have engineered E. coli with decreased content of methionine residues in its proteins and subjected the bacteria to a variety of oxidative stresses. We found that these engineered cells were much more readily killed by the stresses than their wild-type counterparts.[unreadable] [unreadable] Work on the second specific aim has been focused on developing very sensitive assays for iron regulatory protein-2 so that we can study its regulation without the perturbations induced by previous studies utilizing overexpression to facilitate detection. We have also avoided utilizing chelators such as desferoxamine because we discovered that the compound alkalinizes lysosomes, leading to vacuolization and inhibition of unloading of iron from the transferrin-transferrin receptor complex.[unreadable] [unreadable] The third specific aim focused on cryoelectron microspcopy to image the interaction of purified superoxide dismutase mutants with purified 20S proteasome from rat or cow. To date, these efforts have not been successful, perhaps because of the heterogeneity of the mammalian proteasome. We are thus now investigating the much simpler archebacterial proteosomes, which have only a single alpha and beta subunit rather than the seven each present in the mammalian enzyme.